Polypeptides Having Phytase Activity and Polynucleotides Encoding Same

ABSTRACT

The invention relates to a phytase derived from  Citrobacter braakii  and related phytases. The phytases belong to the acid histidine phosphatase family, are acid-stable, of an excellent performance in animal feed, of a high specificity towards the substrate phytate, and expectedly of a high specific activity. The invention also relates to the corresponding DNA, the recombinant and wild-type production of the phytases, as well as the use thereof.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides having phytaseactivity and isolated polynucleotides encoding the polypeptides. Thepolypeptides are related to a phytase derived from Citrobacter braakii,the amino acid sequence of which is shown in the appended sequencelisting as SEQ ID NO: 4. The invention also relates to nucleic acidconstructs, vectors, and host cells comprising the polynucleotides aswell as methods for producing and using the polypeptides, in particularwithin animal feed.

2. Description of the Related Art

Phytases are well-known enzymes, as are the advantages of adding them tofoodstuffs for animals, including humans. Phytases have been isolatedfrom very many sources, including a number of fungal and bacterialstrains.

The acid histidine phosphatase appA of Escherichia coli as well as othergram-negative bacterial phytases are known to have a high specificactivity.

The production by Citrobacter braakii YH-15 of an intracellular phytaseis reported by Kim et al in Biotechnology Letters 25: 1231-1234, 2003.KR-2004-A-045267 and WO-2004/085638 disclose, as SEQ ID NO: 7, the aminoacid sequence of a phytase from Citrobacter braakii YH-15, deposited asKCCM 10427. This amino acid sequence is included herein as SEQ ID NO: 5.WO-2004/085638 was published on Jul. 10, 2004, viz. after the firstpriority date of the present application.

It is an object of the present invention to provide alternativepolypeptides having phytase activity and polynucleotides encoding thepolypeptides. The polypeptides of the invention are preferably ofamended, more preferably improved, properties, for example of adifferent substrate specificity, of a higher specific activity, of anincreased stability (such as acid-stability, heat-stability, and/orprotease stability, in particular pepsin stability), of an amended pHoptimum (such as a lower, or higher pH optimum), and/or of an improvedperformance in animal feed (such as an improved release and/ordegradation of phytate).

SUMMARY OF THE INVENTION

The present invention relates to polypeptides having phytase activity,selected from the group consisting of: (a) a polypeptide having an aminoacid sequence which has at least 98.6% identity with (i) amino acids 1to 411 of SEQ ID NO: 2, and/or (ii) the mature polypeptide part of SEQID NO: 2; (b) a variant comprising a deletion, insertion, and/orconservative substitution of one or more amino acids of (i) amino acids1 to 411 of SEQ ID NO: 2, and/or (ii) the mature polypeptide part of SEQID NO: 2; and/or (c) a fragment of (i) amino acids 1 to 411 of SEQ IDNO: 2, and/or (ii) the mature polypeptide part of SEQ ID NO: 2.

The invention also relates to isolated polynucleotides encoding apolypeptide having phytase activity, selected from the group consistingof: (a) a polynucleotide encoding a polypeptide having an amino acidsequence which has at least 98.6% identity with amino acids 1 to 411 ofSEQ ID NO: 2; and (b) a polynucleotide having at least 98.3% identitywith nucleotides 67 to 1299 of SEQ ID NO: 1.

The invention also relates to nucleic acid constructs, recombinantexpression vectors, and recombinant host cells comprising thepolynucleotides.

The invention also relates to methods for producing such polypeptideshaving phytase activity comprising (a) cultivating a recombinant hostcell comprising a nucleic acid construct comprising a polynucleotideencoding the polypeptide under conditions conducive for production ofthe polypeptide; and (b) recovering the polypeptide.

The invention also relates to methods of using the polypeptides of theinvention in animal feed, as well as animal feed and animal feedadditive compositions containing the polypeptides.

The invention further relates to a nucleic acid construct comprising agene encoding a protein operably linked to a nucleotide sequenceencoding a signal peptide consisting of (i) nucleotides 1 to 66 of SEQID NO: 1 or (ii) nucleotides 1 to 66 of SEQ ID NO: 3; wherein the geneis foreign to the nucleotide sequence.

DEFINITIONS

Phytase activity: In the present context a polypeptide having phytaseactivity (a phytase) is an enzyme which catalyzes the hydrolysis ofphytate (myo-inositol hexakis-phosphate) to (1) myo-inositol and/or (2)mono-, di-, tri-, tetra- and/or penta-phosphates thereof and (3)inorganic phosphate.

The ENZYME site at the internet (http://www.expasy.ch/enzyme/) is arepository of information relative to the nomenclature of enzymes. It isprimarily based on the recommendations of the Nomenclature Committee ofthe International Union of Biochemistry and Molecular Biology (IUB-MB)and it describes each type of characterized enzyme for which an EC(Enzyme Commission) number has been provided (Bairoch A. The ENZYMEdatabase, 2000, Nucleic Acids Res 28:304-305). See also the handbookEnzyme Nomenclature from NC-IUBMB, 1992).

According to the ENZYME site, three different types of phytases areknown: A 3-phytase (myo-inositol hexaphosphate 3-phosphohydrolase, EC3.1.3.8), a 6-phytase (myo-inositol hexaphosphate 6-phosphohydrolase, EC3.1.3.26), and a 5-phytase (EC 3.1.3.72). For the purposes of thepresent invention, all types are included in the definition of phytase.

In a particular embodiment, the phytases of the invention belong to thefamily of acid histidine phosphatases, which includes the Escherichiacoli pH 2.5 acid phosphatase (gene appA) as well as fungal phytases suchas Aspergillus awamorii phytases A and B (EC: 3.1.3.8) (gene phyA andphyB). The histidine acid phosphatases share two regions of sequencesimilarity, each centered around a conserved histidine residue. Thesetwo histidines seem to be involved in the enzymes' catalytic mechanism.The first histidine is located in the N-terminal section and forms aphosphor-histidine intermediate while the second is located in theC-terminal section and possibly acts as proton donor.

In a further particular embodiment, the phytases of the invention have aconserved active site motif, viz. R—H-G-X—R—X—P, wherein X designatesany amino acid (see amino acids 16 to 22 of SEQ ID NOs: 2 and 4).

For the purposes of the present invention the phytase activity isdetermined in the unit of FYT, one FYT being the amount of enzyme thatliberates 1 micro-mol inorganic ortho-phosphate per min. under thefollowing conditions: pH 5.5; temperature 37° C.; substrate: sodiumphytate (C₆H₆O₂₄P₆Na₁₂) in a concentration of 0.0050 mol/l. Suitablephytase assays are the FYT and FTU assays described in Example 1 of WO00/20569. FTU is for determining phytase activity in feed and premix.Phytase activity may also be determined using the phytase assays ofExamples 4, 7 and 8 herein.

The pH-optimum of a polypeptide of the invention is determined byincubating the phytase at various pH-values, using a substrate in apre-determined concentration and a fixed incubation temperature. ThepH-optimum is then determined from a graphical representation of phytaseactivity versus pH. In a particular embodiment, the FYT assay is used,viz. the substrate is 5 mM sodium phytate, the reaction temperature 37°C., and the activity is determined in FYT units at various pH-values,for example pH 2-12, using suitable buffers, such as: 100 mM succinicacid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl₂, 150 mM KCl,0.01% Triton X-100 adjusted to pH-values 2.0, 2.5, 3.0, 3.5, 4.0, 5.0,6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 with HCl or NaOH. In anotherparticular embodiment, the phytase assay of any one of Examples 4, 7 and8 is used, viz. the substrate is 0.5 mM, preferably 5 mM, Na-phytate,which is dissolved in a buffer of the desired pH (such as thosementioned above), and soluble phosphate is determined by complexationwith molybdate/iron and measurement of optical density at 750 nm, or,using the assay of Examples 7 and 8, with molybdate/vanadate andmeasuring absorbancy at 405 nm. Blind (Example 4 test): 20 ul sample,100 ul substrate and 120 ul color reagent is mixed, incubated 5 min at37° C. and OD_(Blind) measured at 750 nm. Sample: 20 ul sample, 100 ulsubstrate is mixed, incubated 30 min at 37° C., 120 ul color reagent isadded, incubated 5 min at 37° C., and OD_(sample) is measured at 750 nm.The phytase activity is measured as OD=OD_(sample)−OD_(Blind). Arelatively low pH-optimum means a pH-optimum below pH 5.0, for examplebelow pH 4.5, 4.0, 3.5, 3.0, 2.5, or even below 2.0. A relatively highpH-optimum means a pH-optimum above pH 5.0, for example above pH 5.5,6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or even above 9.0.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide which is at least 20% pure, preferably at least40% pure, more preferably at least 60% pure, even more preferably atleast 80% pure, most preferably at least 90% pure, and even mostpreferably at least 95% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation which contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, at most 3%, even morepreferably at most 2%, most preferably at most 1%, and even mostpreferably at most 0.5% by weight of other polypeptide material withwhich it is natively associated. It is, therefore, preferred that thesubstantially pure polypeptide is at least 92% pure, preferably at least94% pure, more preferably at least 95% pure, more preferably at least96% pure, more preferably at least 96% pure, more preferably at least97% pure, more preferably at least 98% pure, even more preferably atleast 99%, most preferably at least 99.5% pure, and even most preferably100% pure by weight of the total polypeptide material present in thepreparation.

The polypeptides of the present invention are preferably in asubstantially pure form. In particular, it is preferred that thepolypeptides are in “essentially pure form”, i.e., that the polypeptidepreparation is essentially free of other polypeptide material with whichit is natively associated. This can be accomplished, for example, bypreparing the polypeptide by means of well-known recombinant methods orby classical purification methods.

Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form.”

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences, as well as the degree of identity between twonucleotide sequences, is determined by the program “align” which is aNeedleman-Wunsch alignment (i.e. global alignment), useful for bothprotein and DNA alignments. The default scoring matrix BLOSUM50 and thedefault identity matrix are used for protein and DNA alignmentsrespectively. The penalty for the first residue in a gap is −12 forproteins and −16 for DNA. While the penalties for additional residues ina gap are −2 for proteins and −4 for DNA.

“Align” is part of the FASTA package version v20u6 (see W. R. Pearsonand D. J. Lipman (1988), “Improved Tools for Biological SequenceAnalysis”, PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid andSensitive Sequence Comparison with FASTP and FASTA,” Methods inEnzymology 183:63-98). FASTA protein alignments use the Smith-Watermanalgorithm with no limitation on gap size (see “Smith-Watermanalgorithm”, T. F. Smith and M. S. Waterman (1981) J. Mol. Biol.147:195-197).

The Needleman-Wunsch algorithm is described in Needleman, S. B. andWunsch, C. D., (1970), Journal of Molecular Biology, 48: 443-453, andthe align program by Myers and W. Miller in “Optimal Alignments inLinear Space” CABIOS (computer applications in the biosciences) (1988)4:11-17.

The degree of identity between the target (or sample, or test) sequenceand a specified sequence (e.g. amino acids 1 to 411 of SEQ ID NO: 2) mayalso be determined as follows: The sequences are aligned using theprogram “align.” The number of perfect matches (“N-perfect-match”) inthe alignment is determined (a perfect match means same amino acidresidue in same position of the alignment, usually designated with a “|”in the alignment). The length of the specified sequence (the number ofamino acid residues) is determined (“N-specified”, in the examplementioned above=411). The degree of identity is calculated as the ratiobetween “N-perfect-match” and “N-specified” (for conversion topercentage identity, multiply by 100).

In an alternative embodiment, the degree of identity between a target(or sample, or test) sequence and the specified sequence (e.g. aminoacids 1 to 411 of SEQ ID NO: 2) is determined as follows: The twosequences are aligned using the program “align.” The number of perfectmatches (“N-perfect-match”) in the alignment is determined (a perfectmatch means same amino acid residue in same position of the alignment,usually designated with a “1” in the alignment). The common length ofthe two aligned sequences is also determined, viz. the total number ofamino acids in the overlapping part of the alignment (“N-overlap”). Thedegree of identity is calculated as the ratio between “N-perfect-match”and “N-overlap” (for conversion to percentage identity, multiply by100). In one embodiment, N-overlap includes trailing and leading gapscreated by the alignment, if any. In another embodiment, N-overlapexcludes trailing and leading gaps created by the alignment, if any.

In another alternative embodiment, the degree of identity between atarget (or sample, or test) sequence and a specified sequence (e.g.amino acids 1 to 411 of SEQ ID NO: 2) is determined as follows: Thesequences are aligned using the program “align.” The number of perfectmatches (“N-perfect-match”) in the alignment is determined (a perfectmatch means same amino acid residue in same position of the alignment,usually designated with a “1” in the alignment). The length of thetarget sequence (the number of amino acid residues) is determined(“N-target”). The degree of identity is calculated as the ratio between“N-perfect-match” and “N-target” (for conversion to percentage identity,multiply by 100).

Preferably, the overlap is at least 20% of the specified sequence(“N-overlap” as defined above, divided by the number of the amino acidsin the specified sequence (“N-specified”), and multiplied by 100), morepreferably at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or at least 95%. This means that at least 20%(preferably 25-95%) of the amino acids of the specified sequence end upbeing included in the overlap, when the sample sequence is aligned tothe specified sequence.

In the alternative, the overlap is at least 20% of the target (orsample, or test) sequence (“N-overlap” as defined above, divided by“N-target” as defined above, and multiplied by 100), more preferably atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or at least 95%. This means that at least 20% (preferably 25-95%)of the amino acids of the target sequence end up being included in theoverlap, when aligned against the specified sequence).

Polypeptide Fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more amino acids deleted from the aminoand/or carboxyl terminus of the mature peptide part of the specifiedsequence, e.g. SEQ ID NO: 2 or 4, or a homologous sequence thereof,wherein the fragment has phytase activity. In particular embodiments,the fragment contains at least 350, 360, 370, 380, 390, 400, 405, or atleast 410 amino acid residues.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more nucleotides deleted from the 5′ and/or 3′end of the mature peptide encoding part of the specified sequence, e.g.SEQ ID NO: 1 or 3, or a homologous sequence thereof, wherein thesubsequence encodes a polypeptide fragment having phytase activity. Inparticular embodiments, the subsequence contains at least 1050, 1080,1110, 1140, 1170, 1200, 1215, or at least 1230 nucleotides.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively associated. A substantially pure polynucleotide may, however,include naturally occurring 5′ and 3′ untranslated regions, such aspromoters and terminators. It is preferred that the substantially purepolynucleotide is at least 90% pure, preferably at least 92% pure, morepreferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 97% pure, evenmore preferably at least 98% pure, most preferably at least 99%, andeven most preferably at least 99.5% pure by weight. The polynucleotidesof the present invention are preferably in a substantially pure form. Inparticular, it is preferred that the polynucleotides disclosed hereinare in “essentially pure form”, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively associated. Herein, the term “substantially purepolynucleotide” is synonymous with the terms “isolated polynucleotide”and “polynucleotide in isolated form.” The polynucleotides may be ofgenomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinationsthereof.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term “expression cassette” when the nucleic acid constructcontains the control sequences required for expression of a codingsequence of the present invention.

Control sequence: The term “control sequences” is defined herein toinclude all components, which are necessary or advantageous for theexpression of a polynucleotide encoding a polypeptide of the presentinvention. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG. The codingsequence may a DNA, cDNA, or recombinant nucleotide sequence.

Mature polypeptide part: When used herein the terms “mature polypeptidepart” or “mature peptide part” refer to that part of the polypeptidewhich is secreted by a cell which contains, as part of its geneticequipment, a polynucleotide encoding the polypeptide. In other words,the mature polypeptide part refers to that part of the polypeptide whichremains after the signal peptide part is cleaved off once it hasfulfilled its function of directing the encoded polypeptide into thecell's secretory pathway. The predicted signal peptide part of SEQ IDNOs: 2 and 4 is amino acids −22 to −1 thereof, which means that thepredicted mature polypeptide part of SEQ ID NOs: 2 and 4 corresponds toamino acids 1 to 411 thereof. However, a slight variation may occur fromhost cell to host cell, and therefore the expression mature polypeptidepart is preferred.

Mature polypeptide encoding part: When used herein the term “maturepolypeptide encoding part” or “mature polypeptide coding sequence”refers to that part of the polynucleotide encoding the polypeptide whichencodes the mature polypeptide part. For example, for SEQ ID NO: 1, thepredicted mature polypeptide encoding part corresponds to nucleotides 67to 1299 (encoding amino acids 1 to 411 of SEQ ID NO: 2).

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the invention, and which is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typewhich is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct comprising a polynucleotide ofthe present invention.

Modification: The term “modification” means herein any chemicalmodification of the specified polypeptide, e.g. the polypeptideconsisting of the amino acids 1 to 411 of SEQ ID NO: 2 or 4, as well asgenetic manipulation of the DNA encoding that polypeptide. Themodification(s) can be substitution(s), deletion(s) and/or insertions(s)of the amino acid(s) as well as replacement(s) of amino acid sidechain(s).

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having phytase activity produced by an organismexpressing a modified nucleotide sequence of SEQ ID NO: 1 or 3. Themodified nucleotide sequence is obtained through human intervention bymodification of the nucleotide sequence disclosed in SEQ ID NO: 1 or 3.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having PhytaseActivity

In a first aspect, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofidentity to amino acids 1 to 411 of SEQ ID NO: 2 (i.e., the maturepolypeptide) of at least 98.6%.

In particular embodiments, the degree of identity is at least 98.7%,98.8%, 98.9%, 99%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, or at least 99.9%, which have phytase activity(hereinafter “homologous polypeptides”).

In other particular embodiments, the homologous polypeptides have anamino acid sequence which differs by twenty, eighteen, sixteen,fourteen, twelve, ten, eight, six, five, four, three, two, or by oneamino acid from amino acids 1 to 411 of SEQ ID NO: 2.

In alternative embodiments, the degree of identity to amino acids 1 to411 of SEQ ID NO: 2 (i.e., the mature polypeptide) is at least 70, 80,85, 90, 95, 97, 98%, 98.2%, 98.3%, 98.4%, or 98.5%.

In particular embodiments, the polypeptide of the present inventioncomprises the amino acid sequence of SEQ ID NO: 2, or is an allelicvariant thereof; or a fragment thereof that has phytase activity. Instill further particular embodiments, the polypeptide comprises aminoacids 1 to 411 of SEQ ID NO: 2, or an allelic variant thereof; or afragment thereof that has phytase activity.

In a second aspect, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofidentity to amino acids 1 to 411 of SEQ ID NO: 4 (i.e., the maturepolypeptide) of at least 99.1%.

In particular embodiments, the degree of identity is at least 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9%, which havephytase activity (hereinafter “homologous polypeptides”).

In other particular embodiments, the homologous polypeptides have anamino acid sequence which differs by twenty, eighteen, sixteen,fourteen, twelve, ten, eight, six, five, four, three, two, or by oneamino acid from amino acids 1 to 411 of SEQ ID NO: 4.

In alternative embodiments, the degree of identity to amino acids 1 to411 of SEQ ID NO: 4 (i.e., the mature polypeptide) is at least 70, 80,85, 90, 95, 97, 98%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%,98.9%, or at least 99.0%.

In particular embodiments, the polypeptide of the present inventioncomprises the amino acid sequence of SEQ ID NO: 4, or is an allelicvariant thereof; or a fragment thereof that has phytase activity. Instill further particular embodiments, the polypeptide comprises aminoacids 1 to 411 of SEQ ID NO: 4, or an allelic variant thereof; or afragment thereof that has phytase activity.

In a third aspect, the present invention relates to isolatedpolypeptides having phytase activity which are encoded bypolynucleotides which hybridize under at least medium, preferablymedium, stringency conditions with (i) nucleotides 67 to 1299 of SEQ IDNO: 1, (ii) the mature polypeptide encoding part of SEQ ID NO: 1, and/or(iii) a complementary strand of any one of (i), and (ii), and/or (iv) asubsequence of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T.Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, ColdSpring Harbor, N.Y.). A subsequence of SEQ ID NO: 1 contains at least100 contiguous nucleotides or preferably at least 200 contiguousnucleotides. Moreover, the subsequence may encode a polypeptide fragmentwhich has phytase activity.

In particular embodiments, the hybridization takes place under at leastmedium-high, at least high, or at least very high stringency conditions;preferably under medium-high, high, or very high stringency conditions.

In alternative embodiments, the hybridization is conducted under verylow, or low stringency conditions.

The nucleotide sequence of SEQ ID NO: 1, or a subsequence thereof, aswell as the amino acid sequence of SEQ ID NO: 2, or a fragment thereof,may be used to design a nucleic acid probe to identify and clone DNAencoding polypeptides having phytase activity from strains of differentgenera or species according to methods well known in the art. Inparticular, such probes can be used for hybridization with the genomicor cDNA of the genus or species of interest, following standard Southernblotting procedures, in order to identify and isolate the correspondinggene therein. Such probes can be considerably shorter than the entiresequence, but should be at least 14, preferably at least 25, morepreferably at least 35, and most preferably at least 70 nucleotides inlength. It is, however, preferred that the nucleic acid probe is atleast 100 nucleotides in length. For example, the nucleic acid probe maybe at least 200 nucleotides, preferably at least 300 nucleotides, morepreferably at least 400 nucleotides, or most preferably at least 500nucleotides in length. Even longer probes may be used, e.g., nucleicacid probes which are at least 600 nucleotides, at least preferably atleast 700 nucleotides, more preferably at least 800 nucleotides, or mostpreferably at least 900 nucleotides in length. Both DNA and RNA probescan be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA library prepared from such other organisms may, therefore,be screened for DNA which hybridizes with the probes described above andwhich encodes a polypeptide having phytase activity. Genomic or otherDNA from such other organisms may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA which is homologous with SEQ ID NO: 1,or a subsequence thereof, the carrier material is used in a Southernblot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the nucleotide sequence shown in SEQ ID NO: 1, thecomplementary strand thereof, or a subsequence thereof, under very lowto very high stringency conditions. Molecules to which the nucleic acidprobe hybridizes under these conditions can be detected using X-rayfilm.

In a particular embodiment, the nucleic acid probe is any one of SEQ IDNOs: 1, and 3-8. In another particular embodiment, the nucleic acidprobe is the complementary strand of nucleotides 67 to 450, nucleotides450 to 900, or nucleotides 900 to 1299 of SEQ ID NO: 1. In a furtherparticular embodiment, the nucleic acid probe is a polynucleotidesequence which encodes the polypeptide of SEQ ID NO: 2, or a subsequencethereof. In a still further particular embodiment, the nucleic acidprobe is SEQ ID NO: 1, in particular any one of the mature polypeptidecoding regions thereof.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting 15, procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

Under salt-containing hybridization conditions, the effective T_(m) iswhat controls the degree of identity required between the probe and thefilter bound DNA for successful hybridization. The effective T_(m) maybe determined using the formula below to determine the degree ofidentity required for two DNAs to hybridize under various stringencyconditions.

Effective T_(m)=81.5+16.6(log M[Na⁺])+0.41 (% G+C)−0.72(% formamide)

(See www.ndsu.nodak.edu/instruct/mcclean/plsc731/dna/dna6.htm)

“G+C” designates the content of nucleotides G and T in the probe. Formedium stringency, for example, the formamide is 35% and the Na⁺concentration for 5×SSPE is 0.75 M.

In a fourth aspect, the present invention relates to isolatedpolypeptides having phytase acitivity, and the following physicochemicalproperties (as analyzed on the substantially pure polypeptides):

(i) a high specific activity, such as a specific activity on phytate ofat least 50% of the specific activity of E. coli appA (SPTREMBL:Q8GN88),the specific activity being preferably measured in the units of FYT permg phytase enzyme protein;(ii) acid-stability; such as

(a) at least 60%, preferably at least 65%, at least 70%, or at least75%, residual activity after incubation over night at 37° C. inglycine/hydrochloric acid buffer pH 2.2, relative to the residualactivity after incubation over night at 37° C. in HEPES buffer pH 7.0;

(b) at least 80%, preferably at least 85%, at least 90%, or at least95%, residual activity after incubation over night at 37° C. inglycine/hydrochloric acid buffer pH 3.0, relative to the residualactivity after incubation over night at 37° C. in HEPES buffer pH 7.0;and/or

(c) a residual phytase activity after 2 hours incubation at atemperature of 25, 30, 35, or 37° C., preferably 37° C., and a pH of2.2, 2.4, 2.5, 2.6, 2.8, 3.0, 3.2, 3.4, or 3.5, preferablyglycine/hydrochloric acid buffers of pH 2.2, or 3.0, of at least 50%,compared to the residual activity of E. coli appA (SPTREMBL:Q8GN88);

(iii) heat-stability, such as a residual phytase activity after 0.5, 1,1.5, or 2 hours, preferably 0.5 hours, of incubation at a pH of 5.5 anda temperature of 55, 60, 65, 70, 75, 80, 85 or 95° C., preferably 70°C., of at least 50%, compared to the residual activity of E. coli appA(SPTREMBL:Q8GN88);(iv) protease-stability, such as a residual phytase activity after 0.5,1, 1.5, or 2 hours, preferably 1 hour, incubation at a temperature of20, 25, 30, 35, or 37° C., preferably 37° C., and a pH of 5.5, in thepresence of 0.1 mg/ml pepsin, of at least 50%, compared to the residualactivity of E. coli appA (SPTREMBL:Q8GN88); and/or(v) a pH-optimum below pH 5.0, for example below pH 4.5, 4.0, 3.5, 3.0,2.5, or even below 2.0, determined using the FYT assay, and/or using theassay of Example 4, as described hereinbefore.

In particular embodiments of aspect (i) above, the specific activity isat least 60, 70, 80, 90, 100, 110, 120, 130, 140, or at least 150% ofthe specific activity of E. coli appA. In particular embodiments of eachof aspects (ii) to (iv) above, the residual activity is at least 60, 70,80, 90, 100, 110, 120, 130, 140, or at least 150% of the residualactivity of E. coli appA.

In a fifth aspect, the activity of the enzyme of the invention, at pH5.0 and 37° C., measured on the substrate pNP-phosphate is less than 11%of the activity of the enzyme measured on the substrate phytate,reference being had to Example 7 herein. Preferably, the ratio is lessthan 10%, 9%, 8%, 7%, 6%, or less than 5%. The ratio of pNP to phytatehydrolysis is indicative of the true phytase nature of the enzyme. Ahigh ratio of activity on pNP relative to activity on phytate mayindicate that the enzyme in question is a phosphatase with relativelylow substrate specificity, whereas a low ratio, as is the case for thephytase tested in Example 7, indicates that this is an enzyme morespecifically accepting phytate as a substrate.

In a sixth aspect, the phytase of the invention has a higher release ofphosphorous (P) in the in vitro model of Example 6 herein, as comparedto the phytase from Peniophora lycii, preferably at least 110% thereof,more preferably at least 120%, 130%, or at least 140% thereof. In oneembodiment, the phytase of the invention, dosed 0.25 FYT/g feed,releases at least 150% phosphorous (P), relative to the phosphorousreleased by the phytase from Peniophora lycii, also dosed 0.25 FYT/gfeed, in the in vitro model of Example 6 herein. Preferably, the releaseis at least 155%, 160%, 165%, 170%, 175%, or at least 180%. In anotherembodiment, the phytase of the invention, dosed 0.75 FYT/g feed,releases at least 150% phosphorous (P), relative to the phosphorousreleased by the phytase from Peniophora lycii, also dosed 0.75 FYT/gfeed, in the in vitro model of Example 6 herein. Preferably, the releaseis at least 155%, 160%, 165%, 170%, 175%, 180%, 185%, or at least 190%(see Table 2 in Example 6: 367/190 makes 193).

In a seventh aspect, the phytase of the invention has a residualactivity following incubation at 37° C. and in a 0.1 M Glycine/HClbuffer, pH 2.0, for 4 hours of at least 20%, as compared to the activityat time, t=0, the activity (and the residual activity) being assayed at37° C. and pH 5.5 on 1% (w/v) Na-phytate, using a 0.25 M Na-acetatebuffer pH 5.5, buffer blind subtracted. In preferred embodiments, theresidual activity is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or at least 80%. In another embodiment, the phytase ofthe invention has a residual activity following incubation at 37° C. andin a 0.1M Glycine/HCl buffer, pH 2.5, for 1 day (24 hours) of at least20%, as compared to the activity at time, t=0, the activity (and theresidual activity) being assayed at 37° C. and pH 5.5 on 1% (w/v)Na-phytate, using a 0.25 M Na-acetate buffer pH 5.5, buffer blindsubtracted. In preferred embodiments, the residual activity is at least25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%.

In an eighth aspect, the present invention relates to artificialvariants comprising a conservative substitution, deletion, and/orinsertion of one or more amino acids of SEQ ID NO: 2, or the maturepolypeptide thereof. An insertion can be inside the molecule, and/or atthe N- and/or C-terminal end of the molecule in which case it is alsodesignated extension. Preferably, amino acid changes are of a minornature, that is conservative amino acid substitutions; small deletions,typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain—in other words: Changes that do not significantlyaffect the folding and/or activity of the protein.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine).

Other examples of conservative substitutions are substitutions of the 20standard amino acids with non-standard amino acids (such as4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline,and alpha-methyl serine). Conservative substitutions may also include asubstitution into amino acids that are not encoded by the genetic code,and unnatural amino acids. “Unnatural amino acids” have been modifiedafter protein synthesis, and/or have a chemical structure in their sidechain(s) different from that of the standard amino acids. Unnaturalamino acids can be chemically synthesized, and preferably, arecommercially available, and include pipecolic acid, thiazolidinecarboxylic acid, dehydroproline, 3- and 4-methylproline, and3,3-dimethylproline.

In a particular embodiment, the variant does not comprise all of thefollowing four substitutions in combination: N31D, Q139K, L197F, N316K.In another particular embodiment, the variant does not comprise all ofthe following four substitutions in combination: N31D, N121T, K132T,Q139K.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,phytase activity) to identify amino acid residues that are critical tothe activity of the molecule. See also, Hilton et al., 1996, J. Biol.Chem. 271: 4699-4708. The active site of the enzyme or other biologicalinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction, or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64.The identities of essential amino acids can also be inferred fromanalysis of identities with polypeptides which are related to apolypeptide according to the invention.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman et al., 1991, Biochem. 30:10832-10837; U.S. Pat.No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshireet al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells. Mutagenized DNA molecules thatencode active polypeptides can be recovered from the host cells andrapidly sequenced using standard methods in the art. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

The total number of amino acid substitutions (preferably conservativesubstitutions), deletions and/or insertions in the sequence of aminoacids 1 to 411 of SEQ ID NO: 2 is at most 10, preferably at most 9, morepreferably at most 8, more preferably at most 7, more preferably at most6, more preferably at most 5, more preferably at most 4, even morepreferably at most 3, most preferably at most 2, and even mostpreferably 1.

The total number of amino acid substitutions, deletions and/orinsertions of amino acids 1 to 411 of SEQ ID NO: 2 is 10, preferably 9,more preferably 8, more preferably 7, more preferably at most 6, morepreferably at most 5, more preferably 4, even more preferably 3, mostpreferably 2, and even most preferably 1. In the alternative, the totalnumber of amino acid substitutions (preferably conservativesubstitutions), deletions and/or insertions in the sequence of aminoacids 1 to 411 of SEQ ID NO: 2 is at most 50, 45, 40, 35, 30, 25, 20,19, 18, 17, 16, 15, 14, 13, 12, or at most 11.

In a specific embodiment, the polypeptide of the invention is alow-allergenic variant, designed to invoke a reduced immunologicalresponse when exposed to animals, including man. The term immunologicalresponse is to be understood as any reaction by the immune system of ananimal exposed to the polypeptide. One type of immunological response isan allergic response leading to increased levels of IgE in the exposedanimal. Low-allergenic variants may be prepared using techniques knownin the art. For example the polypeptide may be conjugated with polymermoieties shielding portions or epitopes of the polypeptide involved inan immunological response. Conjugation with polymers may involve invitro chemical coupling of polymer to the polypeptide, e.g. as describedin WO 96/17929, WO98/30682, WO98/35026, and/or WO99/00489. Conjugationmay in addition or alternatively thereto involve in vivo coupling ofpolymers to the polypeptide. Such conjugation may be achieved by geneticengineering of the nucleotide sequence encoding the polypeptide,inserting consensus sequences encoding additional glycosylation sites inthe polypeptide and expressing the polypeptide in a host capable ofglycosylating the polypeptide, see e.g. WO00/26354. Another way ofproviding low-allergenic variants is genetic engineering of thenucleotide sequence encoding the polypeptide so as to cause thepolypeptide to self-oligomerize, effecting that polypeptide monomers mayshield the epitopes of other polypeptide monomers and thereby loweringthe antigenicity of the oligomers. Such products and their preparationis described e.g. in WO96/16177. Epitopes involved in an immunologicalresponse may be identified by various methods such as the phage displaymethod described in WO 00/26230 and WO 01/83559, or the random approachdescribed in EP 561907. Once an epitope has been identified, its aminoacid sequence may be altered to produce altered immunological propertiesof the polypeptide by known gene manipulation techniques such as sitedirected mutagenesis (see e.g. WO 00/26230, WO 00/26354 and/orWO00/22103) and/or conjugation of a polymer may be done in sufficientproximity to the epitope for the polymer to shield the epitope.

Sources of Polypeptides Having Phytase Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

A polypeptide of the present invention may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus polypeptide, or a Streptomycespolypeptide; or a gram negative bacterial polypeptide, e.g., anEscherichia coli, Yersinia, Klebsiella, Citrobacter, or a Pseudomonaspolypeptide. In a particular embodiment, the polypeptide is derived fromProteobacteria, such as Gammaproteobacteria, for exampleEnterobacteriales, such as Enterobacteriaceae.

In a particular aspect, the polypeptide derived from Enterobacteriaceaeis a Citrobacter polypeptide, such as a Citrobacter amalonaticus,Citrobacter braakii, Citrobacter farmeri, Citrobacter freundii,Citrobacter gillenii, Citrobacter intermedius, Citrobacter koseri,Citrobacter murliniae, Citrobacter rodentium, Citrobacter sedlakii,Citrobacter werkmanii, Citrobacter youngae, or Citrobacter speciespolypeptide.

In a more preferred aspect, the polypeptide is a Citrobacter braakiipolypeptide, and most preferably a Citrobacter braakii ATCC 51113polypeptide, e.g., the polypeptide of SEQ ID NO: 4. The specific strainis publicly available from the American Type Culture Collection, ATCC.

A polypeptide of the present invention may also be a fungal polypeptide,such as a yeast polypeptide or a filamentous fungal polypeptide.

Strains of the above microorganisms are readily accessible to the publicin a number of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of another microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques whichare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

Polynucleotides

The present invention also relates to isolated polynucleotides having anucleotide sequence which encodes a polypeptide of the presentinvention. In a preferred aspect, the nucleotide sequence is set forthin any one of SEQ ID NOs: 1 or 3. In another preferred aspect, thenucleotide sequence is the mature polypeptide coding region of any oneof SEQ ID NOs: 1 or 3. The present invention also encompasses nucleotidesequences which encode a polypeptide having the amino acid sequence ofSEQ ID NOs: 2 or 4, or the mature polypeptides thereof, which differfrom SEQ ID NOs: 1 or 3, respectively, by virtue of the degeneracy ofthe genetic code. The present invention also relates to subsequences ofSEQ ID NOs: 1 or 3, which encode fragments of SEQ ID NOs: 2 or 4, thathave phytase activity.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of anyone of SEQ ID NOs: 1 or 3, in which the mutant nucleotide sequenceencodes a polypeptide which consists of amino acids 1 to 411 of SEQ IDNOs: 2 or 4.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Citrobacter, or another or relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the nucleotide sequence.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 (i.e., nucleotides 67 to 1299) of atleast 98.3%, and which encode a polypeptide having phytase activity. Inparticular embodiments, the degree of identity is at least 98.4, 98.5,98.6, 98.7, 98.9, 99, 99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7,99.8, or at least 99.9%. In alternative embodiments, the degree ofidentity is at least 61%, or at least 70%, 75%, 80%, 85%, 90%, 94, 97,98, 98.0, 98.1, 98.2, or at least 98.3%.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 3 (i.e., nucleotides 67 to 1299) of atleast 98.9%, and which encode a polypeptide having phytase activity. Inparticular embodiments, the degree of identity is at least 99.0, 99.1,99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or at least 99.9%. Inalternative embodiments, the degree of identity is at least 61%, or atleast 70%, 75%, 80%, 85%, 90%, 94, 97, 98, 98.0, 98.1, 98.2, 98.3, 98.4,98.5, 98.6, 98.7, or at least 98.8%.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH-optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the polypeptide encoding region of SEQID NO: 1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the polypeptide encoded by the nucleotide sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the polypeptide, or by introduction of nucleotidesubstitutions which may give rise to a different amino acid sequence.For a general description of nucleotide substitution, see, e.g., Ford etal., 1991, Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, Science 244: 1081-1085). In the lattertechnique, mutations are introduced at every positively charged residuein the molecule, and the resultant mutant molecules are tested forphytase activity to identify amino acid residues that are critical tothe activity of the molecule. Sites of substrate-polypeptide interactioncan also be determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labelling (see, e.g., de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, Journal of MolecularBiology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, which hybridize under mediumstringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with (i) nucleotides 67 to1299 of SEQ ID NO: 1, (ii) the mature polypeptide encoding part of SEQID NO: 1, and/or (iii) a complementary strand of any one of (i), and/or(ii); or allelic variants and subsequences thereof (Sambrook et al.,1989, supra), as defined herein. In alternative embodiments thehybridization is conducted under very low, or low, stringencyconditions.

The present invention also relates to isolated polynucleotides obtained,or obtainable, by (a) hybridizing a population of DNA under very low,low, medium, medium-high, high, or very high stringency conditions with(i) nucleotides 67 to 1299 of SEQ ID NO: 1, (ii) the mature polypeptideencoding part of SEQ ID NO: 1, and/or (iii) a complementary strand ofany one of (i), and/or (ii); and (b) isolating the hybridizingpolynucleotide, which encodes a polypeptide having phytase activity.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more control sequences which direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences whichmediate the expression of the polypeptide. The promoter may be anynucleotide sequence which shows transcriptional activity in the hostcell of choice including mutant, truncated, and hybrid promoters, andmay be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichodermareesei endoglucanase III, Trichoderma reesei endoglucanase IV,Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, aswell as the NA2-tpi promoter (a hybrid of the promoters from the genesfor Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase); and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionine (CUP1), Saccharomyces cerevisiae3-phosphoglycerate kinase, and Pichia pastoris alcohol oxidase (AOX1).Other useful promoters for yeast host cells are described by Romanos etal., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra, and by Xiong et al in Journal of AppliedMicrobiology 2005, 98, 418-428.

In a preferred aspect, the signal peptide coding region is nucleotides 1to 66 of SEQ ID NO: 1, which encode amino acids 1 to 22 of SEQ ID NO: 2.In another preferred aspect, the signal peptide coding region isnucleotides 1 to 66 of SEQ ID NO: 3, which encode amino acids 1 to 22 ofSEQ ID NO: 4.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a propolypeptide orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GALL systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleotide sequence encoding the polypeptide at such sites.Alternatively, a nucleotide sequence of the present invention may beexpressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like.

A conditionally essential gene may function as a non-antibioticselectable marker. Non-limiting examples of bacterial conditionallyessential non-antibiotic selectable markers are the dal genes fromBacillus subtilis, Bacillus licheniformis, or other Bacilli, that areonly essential when the bacterium is cultivated in the absence ofD-alanine. Also the genes encoding enzymes involved in the turnover ofUDP-galactose can function as conditionally essential markers in a cellwhen the cell is grown in the presence of galactose or grown in a mediumwhich gives rise to the presence of galactose. Non-limiting examples ofsuch genes are those from B. subtilis or B. licheniformis encodingUTP-dependent phosphorylase (EC 2.7.7.10), UDP-glucose-dependenturidylyltransferase (EC 2.7.7.12), or UDP-galactose epimerase (EC5.1.3.2). Also a xylose isomerase gene such as xylA, of Bacilli can beused as selectable markers in cells grown in minimal medium with xyloseas sole carbon source. The genes necessary for utilizing gluconate,gntK, and gntP can also be used as selectable markers in cells grown inminimal medium with gluconate as sole carbon source. Other examples ofconditionally essential genes are known in the art. Antibioticselectable markers confer antibiotic resistance to such antibiotics asampicillin, kanamycin, chloramphenicol, erythromycin, tetracycline,neomycin, hygromycin or methotrexate.

Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3,TRP1, and URA3. Selectable markers for use in a filamentous fungal hostcell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell are theamdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae andthe bar gene of Streptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of the gene product.An increase in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising apolynucleotide of the present invention is introduced into a host cellso that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular microorganisms are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans andStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred aspect, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, orBacillus subtilis cell. In another preferred aspect, the Bacillus cellis an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Pichia pastoris,Pichia methanolica, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomycesoviformis cell. In another most preferred aspect, the yeast host cell isa Kluyveromyces lactis cell. In another most preferred aspect, the yeasthost cell is a Yarrowia lipolytica cell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,or Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaetechrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris,Trametes villosa, Trametes versicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride strain cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75:1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a cell,which in its wild-type form is capable of producing the polypeptide,under conditions conducive for production of the polypeptide; and (b)recovering the polypeptide. Preferably, the cell is of the genusCitrobacter, and more preferably Citrobacter braakii.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence having atleast one mutation in the mature polypeptide coding region of any one ofSEQ ID NOs: 1 and 3, wherein the mutant nucleotide sequence encodes apolypeptide which consists of amino acids 1 to 411 of any one of SEQ IDNOs: 2 and 4, and (b) recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of a polypeptide product, ordisappearance of an polypeptide substrate. For example, an polypeptideassay may be used to determine the activity of the polypeptide asdescribed herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Transgenic Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleotide sequenceencoding a polypeptide having phytase activity of the present inventionso as to express and produce the polypeptide in recoverable quantities.The polypeptide may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

In a particular embodiment, the polypeptide is targeted to the endospermstorage vacuoles in seeds. This can be obtained by synthesizing it as aprecursor with a suitable signal peptide, see Horvath et al in PNAS,Feb. 15, 2000, vol. 97, no. 4, p. 1914-1919.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot) or engineered variants thereof. Examples of monocot plantsare grasses, such as meadow grass (blue grass, Poa), forage grass suchas Festuca, Lolium, temperate grass, such as Agrostis, and cereals,e.g., wheat, oats, rye, barley, rice, sorghum, triticale (stabilizedhybrid of wheat (Triticum) and rye (Secale), and maize (corn). Examplesof dicot plants are tobacco, legumes, such as sunflower (Helianthus),cotton (Gossypium), lupins, potato, sugar beet, pea, bean and soybean,and cruciferous plants (family Brassicaceae), such as cauliflower, rapeseed, and the closely related model organism Arabidopsis thaliana.Low-phytate plants as described e.g. in U.S. Pat. No. 5,689,054 and U.S.Pat. No. 6,111,168 are examples of engineered plants.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers, as well as the individual tissues comprising these parts,e.g. epidermis, mesophyll, parenchyma, vascular tissues, meristems. Alsospecific plant cell compartments, such as chloroplast, apoplast,mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be aplant part. Furthermore, any plant cell, whatever the tissue origin, isconsidered to be a plant part. Likewise, plant parts such as specifictissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g. embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

Conveniently, the expression construct is a nucleic acid construct whichcomprises a nucleic acid sequence encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleic acid sequence in the plant or plant partof choice. Furthermore, the expression construct may comprise aselectable marker useful for identifying host cells into which theexpression construct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences are determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific cell compartment, tissue or plant partsuch as seeds or leaves. Regulatory sequences are, for example,described by Tague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the following promoters may be used: The35S-CaMV promoter (Franck et al., 1980, Cell 21: 285-294), the maizeubiquitin 1 (Christensen A H, Sharrock R A and Quail 1992. Maizepolyubiquitin genes: structure, thermal perturbation of expression andtranscript splicing, and promoter activity following transfer toprotoplasts by electroporation), or the rice actin 1 promoter (Plant Mo.Biol. 18, 675-689.; Zhang W, McElroy D. and Wu R 1991, Analysis of riceAct1 5′ region activity in transgenic rice plants. Plant Cell 3,1155-1165). Organ-specific promoters may be, for example, a promoterfrom storage sink tissues such as seeds, potato tubers, and fruits(Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or frommetabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol.Biol. 24: 863-878), a seed specific promoter such as the glutelin,prolamin, globulin, or albumin promoter from rice (Wu et al., 1998,Plant and Cell Physiology 39: 885-889), a Vicia faba promoter from thelegumin B4 and the unknown seed protein gene from Vicia faba (Conrad etal., 1998, Journal of Plant Physiology 152: 708-711), a promoter from aseed oil body protein (Chen et al., 1998, Plant and Cell Physiology 39:935-941), the storage protein napA promoter from Brassica napus, or anyother seed specific promoter known in the art, e.g., as described in WO91/14772. Furthermore, the promoter may be a leaf specific promoter suchas the rbcs promoter from rice or tomato (Kyozuka et al., 1993, PlantPhysiology 102: 991-1000, the chlorella virus adenine methyltransferasegene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26:85-93), or the aldP gene promoter from rice (Kagaya et al., 1995,Molecular and General Genetics 248: 668-674), or a wound induciblepromoter such as the potato pin2 promoter (Xu et al., 1993, PlantMolecular Biology 22: 573-588). Likewise, the promoter may be inducibleby abiotic treatments such as temperature, drought or alterations insalinity or inducible by exogenously applied substances that activatethe promoter, e.g. ethanol, oestrogens, plant hormones like ethylene,abscisic acid, gibberellic acid, and/or heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of the polypeptide in the plant. For instance, the promoterenhancer element may be an intron which is placed between the promoterand the nucleotide sequence encoding a polypeptide of the presentinvention. For instance, Xu et al., 1993, supra disclose the use of thefirst intron of the rice actin 1 gene to enhance expression.

Still further, the codon usage may be optimized for the plant species inquestion to improve expression (see Horvath et al referred to above).

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38), andit can also be used for transforming monocots, although othertransformation methods are more often used for these plants. Presently,the method of choice for generating transgenic monocots, supplementingthe Agrobacterium approach, is particle bombardment (microscopic gold ortungsten particles coated with the transforming DNA) of embryonic callior developing embryos (Christou, 1992, Plant Journal 2: 275-281;Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162; Vasil et al.,1992, Bio/Technology 10: 667-674). An alternative method fortransformation of monocots is based on protoplast transformation asdescribed by Omirulleh et al., 1993, Plant Molecular Biology 21:415-428.

Following transformation, the transformants having incorporated thereinthe expression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using e.g.co-transformation with two separate T-DNA constructs or site specificexcision of the selection gene by a specific recombinase.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleic acid sequenceencoding a polypeptide having phytase activity of the present inventionunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Transgenic Animals

The present invention also relates to a transgenic, non-human animal andproducts or elements thereof, examples of which are body fluids such asmilk and blood, organs, flesh, and animal cells. Techniques forexpressing proteins, e.g. in mammalian cells, are known in the art, seee.g. the handbook Protein Expression: A Practical Approach, Higgins andHames (eds), Oxford University Press (1999), and the three otherhandbooks in this series relating to Gene Transcription, RNA processing,and Post-translational Processing. Generally speaking, to prepare atransgenic animal, selected cells of a selected animal are transformedwith a nucleic acid sequence encoding a polypeptide having phytaseactivity of the present invention so as to express and produce thepolypeptide. The polypeptide may be recovered from the animal, e.g. fromthe milk of female animals, or the polypeptide may be expressed to thebenefit of the animal itself, e.g. to assist the animal's digestion.Examples of animals are mentioned below in the section headed AnimalFeed.

To produce a transgenic animal with a view to recovering the polypeptidefrom the milk of the animal, a gene encoding the polypeptide may beinserted into the fertilized eggs of an animal in question, e.g. by useof a transgene expression vector which comprises a suitable milk proteinpromoter, and the gene encoding the polypeptide. The transgeneexpression vector is microinjected into fertilized eggs, and preferablypermanently integrated into the chromosome. Once the egg begins to growand divide, the potential embryo is implanted into a surrogate mother,and animals carrying the transgene are identified. The resulting animalcan then be multiplied by conventional breeding. The polypeptide may bepurified from the animal's milk, see e.g. Meade, H. M. et al (1999):Expression of recombinant proteins in the milk of transgenic animals,Gene expression systems: Using nature for the art of expression. J. M.Fernandez and J. P. Hoeffler (eds.), Academic Press.

In the alternative, in order to produce a transgenic non-human animalthat carries in the genome of its somatic and/or germ cells a nucleicacid sequence including a heterologous transgene construct including atransgene encoding the polypeptide, the transgene may be operably linkedto a first regulatory sequence for salivary gland specific expression ofthe polypeptide, as disclosed in WO 00/064247.

Compositions and Uses

In still further aspects, the present invention relates to compositionscomprising a polypeptide of the present invention, as well as methods ofusing these.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of granulates or microgranulates. The polypeptide to be included inthe composition may be stabilized in accordance with methods known inthe art.

The phytase of the invention can be used for degradation, in anyindustrial context, of, for example, phytate, phytic acid, and/or themono-, di-, tri-, tetra- and/or penta-phosphates of myo-inositol. It iswell known that the phosphate moieties of these compounds chelatesdivalent and trivalent cations such as metal ions, i.a. thenutritionally essential ions of calcium, iron, zinc and magnesium aswell as the trace minerals manganese, copper and molybdenum. Besides,the phytic acid also to a certain extent binds proteins by electrostaticinteraction.

Accordingly, preferred uses of the polypeptides of the invention are inanimal feed preparations (including human food) or in additives for suchpreparations.

In a particular embodiment, the polypeptide of the invention can be usedfor improving the nutritional value of an animal feed. Non-limitingexamples of improving the nutritional value of animal feed (includinghuman food), are: Improving feed digestibility; promoting growth of theanimal; improving feed utilization; improving bio-availability ofproteins; increasing the level of digestible phosphate; improving therelease and/or degradation of phytate; improving bio-availability oftrace minerals; improving bio-availability of macro minerals;eliminating the need for adding supplemental phosphate, trace minerals,and/or macro minerals; and/or improving egg shell quality. Thenutritional value of the feed is therefore increased, and the growthrate and/or weight gain and/or feed conversion (i.e. the weight ofingested feed relative to weight gain) of the animal may be improved.

Furthermore, the polypeptide of the invention can be used for reducingphytate level of manure.

Animals, Animal Feed, and Animal Feed Additives

The term animal includes all animals, including human beings. Examplesof animals are non-ruminants, and ruminants. Ruminant animals include,for example, animals such as sheep, goats, horses, and cattle, e.g. beefcattle, cows, and young calves. In a particular embodiment, the animalis a non-ruminant animal. Non-ruminant animals include mono-gastricanimals, e.g. pigs or swine (including, but not limited to, piglets,growing pigs, and sows); poultry such as turkeys, ducks and chicken(including but not limited to broiler chicks, layers); young calves; andfish (including but not limited to salmon, trout, tilapia, catfish andcarps; and crustaceans (including but not limited to shrimps andprawns).

The term feed or feed composition means any compound, preparation,mixture, or composition suitable for, or intended for intake by ananimal.

In the use according to the invention the polypeptide can be fed to theanimal before, after, or simultaneously with the diet. The latter ispreferred.

In a particular embodiment, the polypeptide, in the form in which it isadded to the feed, or when being included in a feed additive, issubstantially pure. In a particular embodiment it is well-defined. Theterm “well-defined” means that the phytase preparation is at least 50%pure as determined by Size-exclusion chromatography (see Example 12 ofWO 01/58275). In other particular embodiments the phytase preparation isat least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure asdetermined by this method.

A substantially pure, and/or well-defined polypeptide preparation isadvantageous. For instance, it is much easier to dose correctly to thefeed a polypeptide that is essentially free from interfering orcontaminating other polypeptides. The term dose correctly refers inparticular to the objective of obtaining consistent and constantresults, and the capability of optimising dosage based upon the desiredeffect.

For the use in animal feed, however, the phytase polypeptide of theinvention need not be that pure; it may e.g. include other polypeptides,in which case it could be termed a phytase preparation.

The phytase preparation can be (a) added directly to the feed (or useddirectly in a treatment process of proteins), or (b) it can be used inthe production of one or more intermediate compositions such as feedadditives or premixes that is subsequently added to the feed (or used ina treatment process). The degree of purity described above refers to thepurity of the original polypeptide preparation, whether used accordingto (a) or (b) above.

Polypeptide preparations with purities of this order of magnitude are inparticular obtainable using recombinant methods of production, whereasthey are not so easily obtained and also subject to a much higherbatch-to-batch variation when the polypeptide is produced by traditionalfermentation methods.

Such polypeptide preparation may of course be mixed with otherpolypeptides.

The polypeptide can be added to the feed in any form, be it as arelatively pure polypeptide, or in admixture with other componentsintended for addition to animal feed, i.e. in the form of animal feedadditives, such as the so-called pre-mixes for animal feed.

In a further aspect the present invention relates to compositions foruse in animal feed, such as animal feed, and animal feed additives, e.g.premixes.

Apart from the polypeptide of the invention, the animal feed additivesof the invention contain at least one fat-soluble vitamin, and/or atleast one water soluble vitamin, and/or at least one trace mineral. Thefeed additive may also contain at least one macro mineral.

Further, optional, feed-additive ingredients are colouring agents, e.g.carotenoids such as beta-carotene, astaxanthin, and lutein; aromacompounds; stabilisers; antimicrobial peptides; polyunsaturated fattyacids; reactive oxygen generating species; and/or at least one otherpolypeptide selected from amongst phytase (EC 3.1.3.8 or 3.1.3.26);xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase(EC 3.2.1.22); protease (EC 3.4.-.-), phospholipase A1 (EC 3.1.1.32);phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5);phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase suchas, for example, alpha-amylase (EC 3.2.1.1); and/or beta-glucanase (EC3.2.1.4 or EC 3.2.1.6).

In a particular embodiment these other polypeptides are well-defined (asdefined above for phytase preparations).

In a particularly preferred embodiment, the phytase of the inventionhaving a relatively low pH-optimum is combined with at least one phytasehaving a higher pH-optimum. Preferred examples of phytases of higherpH-optimum are Bacillus phytases, such as the phytases from Bacilluslicheniformis and Bacillus subtilis, as well as derivatives, variants,or fragments thereof having phytase activity.

The phytase of the invention may also be combined with other phytases,for example ascomycete phytases such as Aspergillus phytases, forexample derived from Aspergillus ficuum, Aspergillus niger, orAspergillus awamori; or basidiomycete phytases, for example derived fromPeniophora lycii, Agrocybe pediades, Trametes pubescens, or Paxillusinvolutus; or derivatives, fragments or variants thereof which havephytase activity.

Thus, in preferred embodiments of the use in animal feed of theinvention, and in preferred embodiments of the animal feed additive andthe animal feed of the invention, the phytase of the invention iscombined with such phytases.

The above-mentioned ascomycete and basidiomycete phytases, in particularthe RONOZYME P phytase derived from Peniophora lycii as well asderivatives, variants, and fragments thereof, may also be combined withBacillus phytases, in particular the B. licheniformis phytase as well aswith a derivative, fragment or variant thereof, in particular for animalfeed purposes.

Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A,Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin,Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000),Plectasins, and Statins, including the compounds and polypeptidesdisclosed in WO 03/044049 and WO 03/048148, as well as variants orfragments of the above that retain antimicrobial activity.

Examples of antifungal polypeptides (AFP's) are the Aspergillusgiganteus, and Aspergillus niger peptides, as well as variants andfragments thereof which retain antifungal activity, as disclosed in WO94/01459 and WO 02/090384.

Examples of polyunsaturated fatty acids are C18, C20 and C22polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoicacid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of reactive oxygen generating species are chemicals such asperborate, persulphate, or percarbonate; and polypeptides such as anoxidase, an oxygenase or a syntethase.

Usually fat- and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed. Either of thesecomposition types, when enriched with a polypeptide of the invention, isan animal feed additive of the invention.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This isso in particular for premixes.

The following are non-exclusive lists of examples of these components:

Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E,and vitamin K, e.g. vitamin K3.

Examples of water-soluble vitamins are vitamin B12, biotin and choline,vitamin B1, vitamin B2, vitamin B6, niacin, folic acid andpanthothenate, e.g. Ca-D-panthothenate.

Examples of trace minerals are manganese, zinc, iron, copper, iodine,selenium, and cobalt.

Examples of macro minerals are calcium, phosphorus and sodium.

The nutritional requirements of these components (exemplified withpoultry and piglets/pigs) are listed in Table A of WO 01/58275.Nutritional requirement means that these components should be providedin the diet in the concentrations indicated.

In the alternative, the animal feed additive of the invention comprisesat least one of the individual components specified in Table A of WO01/58275. At least one means either of, one or more of, one, or two, orthree, or four and so forth up to all thirteen, or up to all fifteenindividual components. More specifically, this at least one individualcomponent is included in the additive of the invention in such an amountas to provide an in-feed-concentration within the range indicated incolumn four, or column five, or column six of Table A.

The present invention also relates to animal feed compositions. Animalfeed compositions or diets have a relatively high content of protein.Poultry and pig diets can be characterised as indicated in Table B of WO01/58275, columns 2-3. Fish diets can be characterised as indicated incolumn 4 of this Table B. Furthermore such fish diets usually have acrude fat content of 200-310 g/kg.

WO 01/58275 corresponds to U.S. Ser. No. 09/779,334 which is herebyincorporated by reference.

An animal feed composition according to the invention has a crudeprotein content of 50-800 g/kg, and furthermore comprises at least onepolypeptide as claimed herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolisable energy of 10-30 MJ/kg; and/or a content ofcalcium of 0.1-200 g/kg; and/or a content of available phosphorus of0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or acontent of methionine plus cysteine of 0.1-150 g/kg; and/or a content oflysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolisable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO01/58275 (R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25,i.e. Crude protein (g/kg)=N (g/kg)×6.25. The nitrogen content isdetermined by the Kjeldahl method (A.O.A.C., 1984, Official Methods ofAnalysis 14th ed., Association of Official Analytical Chemists,Washington D.C.).

Metabolisable energy can be calculated on the basis of the NRCpublication Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C., pp. 2-6, and the European Table of Energy Values forPoultry Feed-stuffs, Spelderholt centre for poultry research andextension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen& Iooijen bv, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one protein. The protein may be an animal protein,such as meat and bone meal, and/or fish meal; or it may be a vegetableprotein. The term vegetable proteins as used herein refers to anycompound, composition, preparation or mixture that includes at least oneprotein derived from or originating from a vegetable, including modifiedproteins and protein-derivatives. In particular embodiments, the proteincontent of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60%(w/w).

Vegetable proteins may be derived from vegetable protein sources, suchas legumes and cereals, for example materials from plants of thefamilies Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, andPoaceae, such as soy bean meal, lupin meal and rapeseed meal.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g. soybean, lupine,pea, or bean.

In another particular embodiment, the vegetable protein source ismaterial from one or more plants of the family Chenopodiaceae, e.g.beet, sugar beet, spinach or quinoa.

Other examples of vegetable protein sources are rapeseed, sunflowerseed, cotton seed, and cabbage.

Soybean is a preferred vegetable protein source.

Other examples of vegetable protein sources are cereals such as barley,wheat, rye, oat, maize (corn), rice, triticale, and sorghum.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or0-20% whey.

Animal diets can e.g. be manufactured as mash feed (non pelleted) orpelleted feed. Typically, the milled feed-stuffs are mixed andsufficient amounts of essential vitamins and minerals are addedaccording to the specifications for the species in question.Polypeptides can be added as solid or liquid polypeptide formulations.For example, a solid polypeptide formulation is typically added beforeor during the mixing step; and a liquid polypeptide preparation istypically added after the pelleting step. The polypeptide may also beincorporated in a feed additive or premix.

The final polypeptide concentration in the diet is within the range of0.01-200 mg polypeptide protein per kg diet, for example in the range of5-30 mg polypeptide protein per kg animal diet.

The phytase of the invention should of course be applied in an effectiveamount, i.e. in an amount adequate for improving solubilisation and/orimproving nutritional value of feed. It is at present contemplated thatthe polypeptide is administered in one or more of the following amounts(dosage ranges): 0.01-200; 0.01-100; 0.5-100; 1-50; 5-100; 10-100;0.05-50; or 0.10-10—all these ranges being in mg phytase polypeptideprotein per kg feed (ppm).

For determining mg phytase polypeptide protein per kg feed, the phytaseis purified from the feed composition, and the specific activity of thepurified phytase is determined using a relevant assay. The phytaseactivity of the feed composition as such is also determined using thesame assay, and on the basis of these two determinations, the dosage inmg phytase protein per kg feed is calculated.

The same principles apply for determining mg phytase polypeptide proteinin feed additives. Of course, if a sample is available of the phytaseused for preparing the feed additive or the feed, the specific activityis determined from this sample (no need to purify the phytase from thefeed composition or the additive).

Signal Peptide

The present invention also relates to nucleic acid constructs comprisinga gene encoding a protein operably linked to a first nucleotide sequenceconsisting of nucleotides 1 to 66 of any one of SEQ ID NOs: 1 or 3,encoding a signal peptide consisting of amino acids 1 to 22 of any oneof SEQ ID NOs: 2 or 4, wherein the gene is foreign to the firstnucleotide sequences.

The present invention also relates to recombinant expression vectors andrecombinant host cells comprising such nucleic acid constructs.

The present invention also relates to methods for producing a proteincomprising (a) cultivating such a recombinant host cell under conditionssuitable for production of the protein; and (b) recovering the protein.

The first nucleotide sequences may be operably linked to foreign genesindividually with other control sequences or in combination with othercontrol sequences. Such other control sequences are described supra.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides which comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more may be heterologous or native to the host cell.Proteins further include naturally occurring allelic and engineeredvariations of the above mentioned proteins and hybrid proteins.

Preferably, the protein is a hormone or variant thereof, polypeptide,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred aspect, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred aspect, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic polypeptide, peroxidase,phytase, polyphenoloxidase, proteolytic polypeptide, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

VARIOUS EMBODIMENTS

The following are additional embodiments of the present invention. Alsoincluded herein are the corresponding aspects relating to nucleic acidsequences, nucleic acid constructs, recombinant expression vectors,recombinant host cells, methods for production of the polypeptides,transgenic plants and animals, and the various uses, methods of use andfeed compositions/additives, all as claimed.

An isolated polypeptide having phytase activity and a residual activityfollowing incubation at 37° C. and in a 0.1 M Glycine/HCl buffer, pH2.0, for 4 hours of at least 20%, as compared to the activity at time,t=0, the activity being assayed at 37° C. and pH 5.5 on 1% (w/v)Na-phytate, using a 0.25 M Na-acetate buffer pH 5.5, buffer blindsubtracted;

preferably with an identity to i) amino acids 1 to 411 of SEQ ID NO: 4,or ii) amino acids 1 to 411 of SEQ ID NO: 2, of at least 50%, preferablyat least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 98%.

An isolated polypeptide having phytase activity and a residual activityfollowing incubation at 37° C. and in a 0.1 M Glycine/HCl buffer, pH2.5, for 24 hours of at least 20%, as compared to the activity at time,t=0, the activity being assayed at 37° C. and pH 5.5 on 1% (w/v)Na-phytate, using a 0.25 M Na-acetate buffer pH 5.5, buffer blindsubtracted;

preferably with an identity to i) amino acids 1 to 411 of SEQ ID NO: 4,or ii) amino acids 1 to 411 of SEQ ID NO: 2, of at least 50%, preferablyat least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 98%.

An isolated polypeptide having phytase activity, wherein the activity ofthe polypeptide, at pH 5.0 and 37° C., measured on the substratepNP-phosphate is less than 11% of the activity of the polypeptidemeasured on the substrate phytate;

preferably with an identity to i) amino acids 1 to 411 of SEQ ID NO: 4,or ii) amino acids 1 to 411 of SEQ ID NO: 2, of at least 50%, preferablyat least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 98%.

An isolated polypeptide having phytase activity, wherein the polypeptidehas a higher release of phosphorous (P), as compared to the phytase fromPeniophora lycii;

preferably as measured in the in vitro model of Example 6 herein;

and/or, wherein the polypeptide preferably has an identity to i) aminoacids 1 to 411 of SEQ ID NO: 4, or ii) amino acids 1 to 411 of SEQ IDNO: 2, of at least 50%, preferably at least 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or at least 98%.

An isolated polypeptide having phytase activity, wherein thepolypeptide, dosed 0.25 FYT/g feed, releases at least 150% phosphorous(P), relative to the phosphorous released by the phytase from Peniophoralycii, also dosed 0.25 FYT/g feed;

preferably as measured in the in vitro model of Example 6 herein;

and/or, wherein the polypeptide preferably has an identity to i) aminoacids 1 to 411 of SEQ ID NO: 4, or ii) amino acids 1 to 411 of SEQ IDNO: 2, of at least 50%, preferably at least 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or at least 98%.

An isolated polypeptide having phytase activity, wherein thepolypeptide, dosed 0.75 FYT/g feed, releases at least 150% phosphorous(P), relative to the phosphorous released by the phytase from Peniophoralycii, also dosed 0.75 FYT/g feed;

preferably as measured in the in vitro model of Example 6 herein;

and/or, wherein the polypeptide preferably has an identity to i) aminoacids 1 to 411 of SEQ ID NO: 4, or ii) amino acids 1 to 411 of SEQ IDNO: 2, of at least 50%, preferably at least 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or at least 98%.

I. An isolated polypeptide having phytase activity, selected from thegroup consisting of: (a) a polypeptide having an amino acid sequencewhich has at least 98.2% identity with (i) amino acids 1 to 411 of SEQID NO: 2, and/or (ii) the mature polypeptide part of SEQ ID NO: 2, (b) apolypeptide which is encoded by a polynucleotide which hybridizes underat least medium stringency conditions with (i) nucleotides 67 to 1299 ofSEQ ID NO: 1, (ii) the mature polypeptide encoding part of SEQ ID NO: 1,and/or (iii) a complementary strand of any one of (i), or (ii); (c) avariant of any one of the polypeptides of (a)(i)-(a)(ii), comprising aconservative substitution, deletion, and/or insertion of one or moreamino acids; and (d) a fragment of any one of the polypeptides of(a)(i)-(a)(ii).

II. An isolated polynucleotide comprising a nucleotide sequence whichencodes the polypeptide of section 1.

III. An isolated polynucleotide encoding a polypeptide having phytaseactivity, selected from the group consisting of: (a) a polynucleotideencoding a polypeptide having an amino acid sequence which has at least98.2% identity with amino acids 1 to 411 of SEQ ID NO: 2; (b) apolynucleotide having at least 95% identity with nucleotides 67 to 1299of SEQ ID NO: 1; and (c) a polynucleotide which hybridizes under atleast medium stringency conditions with (i) nucleotides 67 to 1299 ofSEQ ID NO: 1, (ii) the mature polypeptide encoding part of SEQ ID NO: 1,(iii) a complementary strand of any one of (i), or (ii).

IV. The isolated polynucleotide of any one of sections 11 and III,having at least one mutation in the mature polypeptide coding sequenceof SEQ ID NO: 1, in which the mutant nucleotide sequence encodes apolypeptide comprising amino acids 1 to 411 of SEQ ID NO: 2.

V. A nucleic acid construct comprising the polynucleotide of any one ofsections II-IV operably linked to one or more control sequences thatdirect the production of the polypeptide in an expression host.

VI. A recombinant expression vector comprising the nucleic acidconstruct of section V.

VII. A recombinant host cell comprising the nucleic acid construct ofsection V.

VII. A method for producing the polypeptide of section I comprising (a)cultivating a cell, which in its wild-type form is capable of producingthe polypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

IX. A method for producing the polypeptide of section I comprising (a)cultivating a recombinant host cell comprising a nucleic acid constructcomprising a nucleotide sequence encoding the polypeptide underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

X. A transgenic plant, plant part or plant cell, which has beentransformed with a polynucleotide encoding the polypeptide of section I.

XI. A transgenic, non-human animal, or products, or elements thereof,being capable of expressing the polypeptide of section I.

XII. Use of at least one polypeptide of section I in animal feed.

XIII. Use of at least one polypeptide of section I in the preparation ofa composition for use in animal feed.

XIV. A method for improving the nutritional value of an animal feed,wherein at least one polypeptide of section I is added to the feed.

XV. An animal feed additive comprising (a) at least one polypeptide ofsection I; and (b) at least one fat soluble vitamin, (c) at least onewater soluble vitamin, and/or (d) at least one trace mineral.

XVI. The animal feed additive of section XV, which further comprises atleast one amylase, at least one additional phytase, at least onexylanase, at least one galactanase, at least one alpha-galactosidase, atleast one protease, at least one phospholipase, and/or at least onebeta-glucanase.

XVII. The animal feed additive of section XVI, wherein the additionalphytase has a pH-optimum which is higher than the pH-optimum of thepolypeptide having the amino acid sequence of amino acids 1 to 411 ofSEQ ID NO: 2.

IIXX. An animal feed composition having a crude protein content of 50 to800 g/kg and comprising at least one polypeptide of section I.

A polypeptide having phytase activity selected from the group consistingof:

(a) a polypeptide comprising an amino acid sequence which has at least99.1% identitywith (i) amino acids 1 to 411 of SEQ ID NO: 4, and/or

(ii) the mature polypeptide part of SEQ ID NO: 4;

(b) a variant of any one of the polypeptides of (a)(i)-(a)(ii),comprising a deletion, insertion, and/or conservative substitution ofone or more amino acids; and(c) a fragment of any one of the polypeptides of (a)(i)-(a)(ii).

A polynucleotide encoding a polypeptide having phytase activity,selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide having an amino acidsequence which has at least 99.1% identity with amino acids 1 to 411 ofSEQ ID NO: 4; and(b) a polynucleotide having at least 98.9% identity with nucleotides 67to 1299 of SEQ ID NO: 3.

A polypeptide having phytase activity which comprises, preferably has,an amino acid sequence which has at least 98.6% identity with aminoacids 1 to 411 of SEQ ID NO: 2.

A polypeptide having phytase activity which comprises, preferably has,an amino acid sequence which has at least 99.1% identity with aminoacids 1-411 of SEQ ID NO: 4. Uden identitet, men med variant og fragment

A polypeptide having phytase activity which comprises, preferably has,the sequence of

(i) amino acids 1 to 411 of SEQ ID NO: 2, and/or(ii) the mature polypeptide part of SEQ ID NO: 2; or which polypeptide(a) is a variant of any one of the polypeptides of (i)-(ii), comprisinga deletion, insertion, and/or conservative substitution of one or moreamino acids; or(c) is a fragment of any one of the polypeptides of (i)-(ii).

A polypeptide having phytase activity which comprises, preferably has,the sequence of

(i) amino acids 1 to 411 of SEQ ID NO: 4, and/or(ii) the mature polypeptide part of SEQ ID NO: 4; or which polypeptide(a) is a variant of any one of the polypeptides of (i)-(ii), comprisinga deletion, insertion, and/or conservative substitution of one or moreamino acids; or(c) is a fragment of any one of the polypeptides of (i)-(ii).

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES Example 1 Cloning of a Citrobacter braakii Phytase

A multiple alignment was made of the following acid histidinephosphatases: appA Escherichia coli (SPTREMBL:Q8GN88), phyk Klebsiellaterrigena (SPTREMBL:Q7WSY1), and ypo1648 Yersinia pestis C092(SPTREMBL:Q8ZFP6). Two degenerate oligonucleotide primers were designedon the basis of consensus sequences: 5′-TGG TGA TTG TGT CCC GTC AYG GNGTNM G-3′ (SEQ ID NO: 6, forward primer) 5′-GCC CGG CGG GGT RTT RTCNGG-3′ (SEQ ID NO: 7, reverse primer).

The primers were used for PCR screening of a number of bacterial speciesat annealing temperatures of 45, 48 and 50° C.

A partial phytase gene in the form of a 900 bp DNA fragment wasidentified in Citrobacter braakii ATCC 51113.

The PCR fragment was cloned into the pEZSeq blunt cloning kit (catalogueno. 40501-1 from Lucigen Corporation, 2120 West Greenview Dr., Ste 9,Middleton, Wis. 53562, US). First, the PCR fragment was treated with thePCRTerminator End Repair Kit (part of the pEZSeq blunt cloning kit),which contains a mixture of enzyme activities that has been optimized tocreate blunt, 5′-phosphorylated ends on any type of PCR product. Aftercloning into the pEZSeq vector, the clone was sequenced using twospecific vector primers. By translation of the nucleotide sequence, itwas confirmed that the cloned DNA fragment was part of phytase gene.

For obtaining the full length nucleotide sequence of the gene the DNAWalking SpeedUp Kit (DWSK-V102 from Seegene, Inc., 2nd Fl., MyungjiBldg., 142-21, Samsung-dong, Kangnam-gu, Seoul, 135-090, Korea) wasused, which is designed to capture unknown target sites. For thispurpose, 4 specific oligonucleotides were designed and used with thekit.

TSP1N: 5′- ACATTTTGGTGCTAACCCAGCC-3′ (SEQ ID NO: 8) TSP1C: 5′-AGAAGTTGCCCGTAGTAGGGCC-3′ (SEQ ID NO: 9) TSP2N: 5′-ATTCAGAAACAAGTTCTCCCCCACG-3′ (SEQ ID NO: 10) TSP2C: 5′-ACCAATCTTGCAAATTTAAGCGGGG-3′ (SEQ ID NO: 11)

The correct full length nucleotide sequence encoding the phytase fromCitrobacter braakii ATCC 51113 is shown in the sequence listing as SEQID NO: 3, and the corresponding encoded amino acid sequence has SEQ IDNO: 4. The first 22 amino acids of SEQ ID NO: 4 are expected to be asignal peptide (predicted by Signal P V3.0).

The Citrobacter braakii ATCC 51113 phytase gene was cloned into thepET-30a(+) E. coli expression vector without fusion tags (catalogue no.69909 from Novagen, commercially available from Bie & Berntsen A/S, 7Sandbaekvej, DK-2610 Roedovre, Denmark). In this system, the expressionof the gene is induced by providing a source of T7 RNA polymerase in theE. coli BL21star(DE)pLysS host strain (catalogue no. 69388 from Novagen,commercially available from Bie & Berntsen) which contains a chromosomalcopy of the T7 RNA polymerase gene under the control of the lacUV5promoter. The induction of the target gene was performed by addinglactose to the media. Lactose will bind to the repressor and induce itsdissociation from the operator, permitting transcription from thepromoter.

For expression of the phytase gene, a single colony of the transformedE. coli strain was transferred into an inoculum culture in non-inducingmedia (containing glucose as the sole carbon source) that does notpermit expression of the T7 RNA polymerase. As a negative control E.coli (BL21star(DE)pLysS) containing an empty pET-30(+) vector was used.A small aliquot (approximately 150 micro liter) of the inoculum culturewas transferred into flasks containing lactose as the sole carbonsource. The induction culture was grown overnight with shaking at 300rpm at 37° C.

The cells were harvested by centrifugation and 15 micro liter aliquotsof the supernatant was analysed by SDS-PAGE. As a molecular weight (MW)marker 10 micro liter of the Precision Plus protein standard was used(catalogue no. 161-0363, commercially available from Bio-RadLaboratories Headquarters, 1000 Alfred Nobel Drive, Hercules, Calif.94547, US). A distinct band of MW of approximately 50 kDa was identifiedin the supernatant from the recombinant E. coli strain, but not in thenegative control.

The harvested cell pellet was lysed and the soluble intracellularfraction was also analysed by SDS-PAGE as described above. Also here aband at MW 50 kDa appeared.

This is evidence that the recombinant phytase protein is partiallysecreted to the media. However, a pool of the enzyme still remains inthe intracellular fraction.

The phytase activity of the supernatant and the intracellular fractionwas confirmed by use of the assay of Example 4.

Example 2 Preparation of a Citrobacter braakii Phytase Preparation

Citrobacter braakii ATCC 51113 was grown overnight with shaking (225rpm) at 30° C. in LB medium (25 g of LB Bouillon, Merck 0285,ion-exchanged water ad 1000 ml) with addition of 0.1% (w/w) sodiumphytate. The cells were harvested by centrifugation (4000 rpm, 60 min)and the supernatant discarded. The cell pellet was re-suspended in twovolumes of distilled water with 100 mg/ml lysozyme and lysed byovernight incubation at 37° C. The lysed cells were centrifuged (4000rpm, 2 h) and the supernatant saved and used for acid stabilityanalysis.

Example 3 Acid Stability of the Citrobacter braakii Phytase

50 micro liter of the lysate obtained in Example 2 was mixed with 50micro liter of 100 mM buffers with pH values of 2.2, 3.0(glycine/hydrochloric acid) and 7.0 (HEPES) respectively. The sampleswere incubated over night at 37° C. and analysed for residual phytaseactivity using the analytical procedure described in Example 4. Theresidual phytase activity, expressed as the optical density, is shown inTable 1 below. Furthermore, the activity is calculated in percentrelative to the residual activity at pH 7.

TABLE 1 Residual Activity [OD] after incubation at pH: pH 2.2 - pH 3.0 -pH 7.0 - rela- rela- rela- tive to tive to tive to Strain: pH 2.2 pH 7.0pH 3.0 pH 7.0 pH 7.0 pH 7.0 Citrobacter 0.35 95 0.43 116 0.37 100braakii ATCC 51113

Example 4 Phytase Assay

The assay is based on determination of soluble phosphate by complexationwith molybdate/iron and photometric measurement of the blue color inmicrotiter plates.

The substrate is 0.5 mM Na-phytate (Sigma, P-8810) dissolved in 0.1 Macetate-buffer, pH=5.5. In a particular embodiment the substrateconcentration is 5 mM.

The color reagent is prepared as follows: 1% Ammoniummolybdat (Merck1181, (NH₄)₆Mo₇O₂₄, 4H₂O) is dissolved in 3.2% sulfuric acid (Merck731). 1.1 g ferrosulfate (Merck 3965) is dissolved in 15 ml of the abovemolybdate reagent and 10 ml of 0.5 M sulfuric acid is added. Is freshlyprepared every day, and stored in the dark.

Blind: 20 ul sample, 100 ul substrate and 120 ul color reagent is mixed,incubated 5 min at 37° C. and OD_(Blind) measured at 750 nm.

Sample: 20 ul sample, 100 ul substrate is mixed, incubated 30 min at 37°C., 120 ul color reagent is added, incubated 5 min at 37° C., andOD_(sample) is measured at 750 nm.

OD=OD _(sample) −OD _(Blind).

Example 5 Preparation of Recombinant Phytase

The phytase of SEQ ID NO: 4 was expressed in Bacillus subtilis andpurified using conventional methods: Centrifugation, germ filtration,ammonium sulphate precipitation (80% ammonium sulphate saturation),centrifugation, re-suspension of pellets in buffer A (50 mM sodiumacetate, 1.5 M ammonium sulphate pH 4.5), filtration, hydrophobicinteraction chromatography (Phenyl Toyopearl, loading with buffer A,eluting with buffer B (50 mM sodium acetate pH 4.5)), and cationexchange chromatography (SP-sepharose, loading with 10 mM sodium citratepH 4.0, eluting with a linear salt gradient (10 mM sodium citrate pH4.0+1 M NaCl).

A coomassie-stained SDS-PAGE gel showed the purified mature phytase tobe more than 50% pure on a protein-basis. However most of thenon-phytase bands were found to be degradation products or truncatedforms of the C. braakii phytase. Accordingly the purity was well above80%, when expressed as the amount of phytase and phytase-degradationproducts relative to the total amount of proteins.

Example 6 Performance in Animal Feed

The performance in animal feed of the purified Citrobacter braakiiphytase of Example 5 was compared, in an in vitro model, with theperformance of a commercial phytase from Peniophora lycii described inWO 98/28408, commercially available from DSM Nutritional Products, asthe RONOZYME P phytase. The in vitro model simulates digestion in amonogastric animal and correlates well with results obtained in animaltrials in vivo.

Feed samples composed of 30% soybean meal and 70% maize meal with addedCaCl₂ to a concentration of 5 g calcium per kg feed were prepared andpre-incubated at 40° C. and pH 3.0 for 30 minutes followed by additionof pepsin (3000 U/g feed) and two different dosages of the two phytases,viz. 0.25 or 0.75 phytase units (FYT)/g feed. A blank with no phytaseactivity was also included. The samples were incubated at 40° C. and pH3.0 for 60 minutes followed by pH 4.0 for 30 minutes.

The reactions were stopped and phytic acid and inositol-phosphatesextracted by addition of HCl to a final concentration of 0.5 M andincubation at 40° C. for 2 hours, followed by one freeze-thaw cycle and1 hour incubation at 40° C.

Phytic acid and inositol-phosphates were separated by high performanceion chromatography as described by “Chen, Q. C. and Li, B. W. (2003).Separation of phytic acid and other related inositol phosphates byhigh-performance ion chromatography and its applications. Journal ofChromatography A 1018, 41-52” and quantified according to “Skoglund, E.,Carlsson, N. G., and Sandberg, A. S. (1997). Determination of isomers ofinositol mono- to hexa-phosphates in selected foods and intestinalcontents using high-performance ion chromatography. J. Agric. Food Chem.45, 431-436”.

Released phosphorous was calculated as the difference ininositol-phosphate bound phosphorous (IP-P) between phytase-treated andnon-treated samples.

From the results shown in Table 2 below it is clear that the C. braakiiphytase of the invention is much more effective than the commercialphytase in releasing phosphate from the feed.

TABLE 2 Treatment Dosage (FYT/g) Relative P release (%) P. lycii phytase0.25 100 P. lycii phytase 0.75 190 C. braaki phytase 0.25 184 C. braakiiphytase 0.75 367

Example 7 Substrate Specificity

The activity at pH 5.0 and 37° C. of the purified Citrobacter braakiiphytase of Example 5 was tested on two substrates, viz. phytate andp-nitrophenyl phosphate (pNP-phosphate). More in particular, theactivity on pNP-phosphate relative to the activity on phytate wasdetermined by comparing assay readouts from each substrate to phosphatestandard curves (buffer blind subtracted).

Materials

Enzyme dilution buffer: 0.25 M Na-acetate buffer, pH 5.0 incl. 0.005%Tween-20

Phytase substrate: Sodium phytate from rice (Aldrich 274321) 10 mg/ml in0.25 M Na-acetate buffer pH 5.0

pNP-phosphate substrate: Two 5 mg p-nitrophenyl phosphate tablets (SigmaN9389) dissolved in 10 ml 0.1 M Na-acetate buffer pH 5

Molybdate solution (10% Ammonium hepta-molybdate in 0.25% ammoniasolution):

10 g ammonium hepta-molypdate (Merck 1.001182) dissolved in 90 mlde-ionised H₂O

1 ml 25% ammonia solution (Merck 1.05432)

Vol. adjusted to 100 ml with de-ionised H₂O

Ammonium mono-vanadate reagent (0.24% (w/v) NH₄VO₃ solution in 3.25%HNO₃, Bie & Berntsen, Denmark, LAB17650).

Stop reagent (Molybdate/vanadate reagent in HNO₃): 10 ml molybdatesolution+10 ml ammonium mono-vanadate reagent+20 ml 21.7% nitric acid

Procedure

75 μl/well enzyme solution (or buffer blind) was dispensed in amicrotiter plate (Nunc 269620). 75 μl substrate (sodium phytate orpNP-phosphate) was added and the plate was sealed with a piece ofadhesive plate sealer. The plate was quickly placed in an EppendorffThermomixer equipped with an microtiter plate holder and shaken with 750rpm at 37° C. for 15 min. 75 μl stop reagent was added. Absorbance at405 nm was measured in a microtiter plate spectrophotometer (MolecularDevices Spectramax 384 Plus). As it is well-known for the person skilledin the art, various dilutions of the enzyme were tested in order toobtain suitable absorbance readings at 405 nm.

Results

The phosphate hydrolysis of pNP-phosphate was found to be 4% of that ofphytate. Table 6 of KR-2004-A-045267 (WO-2004/085638) reports theactivity of the YH-15 phytase on pNP-phosphate to be 11.27% relative tothe activity on phytate, which means that the Citrobacter braakiiphytase of the present invention is different in this respect.

Example 8 Acid-Stability

The acid-stability of the purified Citrobacter braakii phytase ofExample 5 was determined as residual phosphate hydrolysis activityfollowing incubation at 37° C. and pH 2.0, 2.5 or 3.0 (0.1M Glycine/HClbuffer). The residual activity on Na-phytate was assayed at pH 5.5, 37°C., buffer blind subtracted, and the results compared to the activity attime, t=0.

Materials

Enzyme dilution buffer: 0.25 M Na-acetate buffer pH 5.5 incl. 0.005%Tween-20

Phytase substrate: 1% (w/v) Na-phytate (Aldrich 274321) dissolved in0.25 M Na-acetate pH 5.5

Stop reagent:10 ml 10% (w/v) (NH₄)₆Mo₇O₂₄4H₂O solution10 ml 0.24% (w/v) NH₄VO₃ solution20 ml 21.7% HNO₃ solution10% (w/v) (NH₄)₆Mo₇O₂₄.4H₂O solution:10 g (NH₄)₆Mo₇O₂₄ 4H₂O (Merck 1.001182) dissolved in 90 ml de-ionisedwater1 ml 25% (w/v) NH₃ solution (Merck 1.05432)Volume adjusted to 100 ml using de-ionised water0.24% (w/v) NH₄VO₃ solution in 3.25% HNO₃ (Bie & Berntsen, Denmark,LAB17650).

A purified stock solution of Citrobacter braakii phytase stored in 20 mMNaAc pH 4.0 was diluted using 0.1M Glycine/HCl buffer (sufficientdilution to ensure that the intended pH value is obtained) and incubatedon an Eppendorf Thermomixer at 37° C., 750 rpm (1.5 ml Eppendorf ProteinLoBind Tube, PCR clean, cat. no. 2243108-1). Residual activity wasassayed employing the procedure described below.

The phytase activity per ml of the phytase stock solution shouldallow 1) dilution using the 0.1 M Glycine/HCl buffer solution in orderto obtain the desired pH, followed by 2) dilution using enzyme dilutionbuffer (see above) in order to obtain the pH of the assay conditions,thereby resulting in a suitable absorbance reading at 405 nm.

Assay Procedure

After time, t=x hours, a sample was withdrawn from each pH incubationmixture and diluted using enzyme dilution buffer (sufficient dilution toensure that a pH of 5.5 is obtained). 75 μl enzyme solution (or bufferblind, consisting of enzyme dilution buffer) was added to wells in amicrotiter plate (Nunc 269620). 75 μl substrate solution was added, theplate sealed with adhesive plate sealer, followed by quick transfer toan Eppendorf Thermomixer equipped with a microtiter plate holder. Theplate was incubated at 37° C. with shaking (750 rpm) for 15 min. 75 μlstop reagent was added and the absorbance read at 405 nm in a microtiterplate spectrophotometer (Molecular Devices Spectramax 190).

Results

A significant residual activity was observed after incubation at pH 2.0,37° C. for 4 hours. Likewise, a significant residual activity wasobserved after 1 day of incubation at pH 2.5, 37° C.

Example 4-2 of KR-2004-A-045267 (see the bottom of p. 27 ofWO-2004/085638) explains that the enzyme activity of the YH-15 phytasewas almost lost after incubation under pH 3.0 for 4 hours. The KRapplication is silent about the buffer used, but from the relatedpublication by Kim et al (Biotechnology Letters 25: 1231-1234, 2003), itappears from FIG. 2that a glycine/HCl buffer was used, and this bufferwas therefore also used in the present example.

In conclusion, the Citrobacter braakii phytase of the present inventionis much more acid-stable as compared to the YH-15 phytase.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. An isolated polypeptide having phytase activity, selected from thegroup consisting of; (a) a polypeptide comprising an amino acid sequencewhich has at least 98.6% identity with (i) amino acids 1 to 411 of SEQID NO: 2, and/or (ii) the mature polypeptide part of SEQ ID NO: 2; (b) avariant of any one of the polypeptides of (a)(i)-(a)(ii), comprising adeletion, insertion, and/or conservative substitution of one or moreamino acids; and (c) a fragment of any one of the polypeptides of(a)(i)-(a)(ii).
 2. The polypeptide of claim 1 selected from the groupconsisting of: (a) a polypeptide comprising an amino acid sequence whichhas at least 99.1% identity with (i) amino acids 1 to 411 of SEQ ID NO:4, and/or (ii) the mature polypeptide part of SEQ ID NO: 4; (b) avariant of any one of the polypeptides of (a)(i)-(a)(ii), comprising adeletion, insertion, and/or conservative substitution of one or moreamino acids; and (c) a fragment of any one of the polypeptides of(a)(i)-(a)(ii).
 3. An isolated polynucleotide comprising a nucleotidesequence which encodes the polypeptide of claim
 1. 4. An isolatedpolynucleotide encoding a polypeptide having phytase activity, selectedfrom the group consisting of: (a) a polynucleotide encoding apolypeptide having an amino acid sequence which has at least 98.6%identity with amino acids 1 to 411 of SEQ ID NO: 2; and (b) apolynucleotide having at least 98.3% identity with nucleotides 67 to1299 of SEQ ID NO:
 1. 5. The polynucleotide of claim 4, selected fromthe group consisting of: (a) a polynucleotide encoding a polypeptidehaving an amino acid sequence which has at least 99.1% identity withamino acids 1 to 411 of SEQ ID NO: 4; and (b) a polynucleotide having atleast 98.9% identity with nucleotides 67 to 1299 of SEQ ID NO:
 3. 6. Anucleic acid construct comprising the polynucleotide of claim 4 operablylinked to one or more control sequences that direct the production ofthe polypeptide in an expression host.
 7. A recombinant expressionvector comprising the nucleic acid construct of claim
 6. 8. Arecombinant host cell comprising the nucleic acid construct of claim 6.9. A method for producing the polypeptide of claim 1 comprising (a)cultivating a cell, which in its wild-type form is capable of producingthe polypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.
 10. A method forproducing the polypeptide of claim 1 comprising (a) cultivating arecombinant host cell comprising a nucleic acid construct comprising anucleotide sequence encoding the polypeptide under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.11. A transgenic plant, plant part or plant cell, which has beentransformed with a polynucleotide encoding the polypeptide of claim 1any on
 12. A transgenic, non-human animal, or products, or elementsthereof, which is capable of expressing the polypeptide of claim 1 anyone of m. 13-14. (canceled)
 15. A method for improving the nutritionalvalue of an animal feed, comprising adding at least one polypeptide ofclaim 1 to the feed.
 16. An animal feed additive comprising (a) at leastone polypeptide of claim 1; and (b) at least one fat soluble vitamin,(c) at least one water soluble vitamin, and/or (d) at least one tracemineral.
 17. The animal feed additive of claim 16, which furthercomprises at least one amylase, at least one additional phytase, atleast one xylanase, at least one galactanase, at least onealpha-galactosidase, at least one protease, at least one phospholipase,and/or at least one beta-glucanase.
 18. An animal feed compositionhaving a crude protein content of 50 to 800 g/kg and comprising at leastone polypeptide of claim 1.